ABSTRACT The need of an effective therapy for metastatic triple negative breast cancer (TNBC) has prompted us to develop a novel combinatorial immunotherapeutic strategy for this very aggressive subtype of breast cancer. In the design of our strategy we have been guided by the realization that to be effective, a therapy has to eradicate both differentiated TNBC cells and TNBC cancer initiating cells (CICs). According to the cancer stem cell theory, CICs play a major role in disease recurrence and metastatic spread, the major causes of patient morbidity and mortality. Furthermore, immunotherapy has to counteract the multiple escape mechanisms utilized by TNBC cells to avoid immune destruction. Therefore, i) to eliminate both differentiated TNBC cells and TNBC CICs, we have selected as a target chondroitin sulphate proteoglycan 4 (CSPG4), since this TA can mediate the immune destruction of both types of targets. In addition, CSPG4 is selectively up-regulated on activated pericytes in the tumor microenvironment. Therefore, CSPG4 immunotargeting inhibits neoangiogenesis and contributes to the elimination of TNBC cells, even those with CSPG4 loss or down regulation. Lastly CSPG4 has a restricted distribution in normal tissues. Therefore, CSPG4 immunotargeting is not expected to cause side effects because of the targeting of normal tissues. ii) To counteract the escape mechanisms caused by abnormalities in HLA class I antigen processing machinery in TNBC cells, we have selected as effectors NK cells which do not require HLA class I antigen expression for recognition of target cells. To activate and expand the NK cell effector population at the level of the immune synapse, we have added IL-15 to the conventional bispecific NK Cell immune engagers (BiKEs) platform and used it to crosslink the scFv fragments derived from the CSPG4-specific mAb 763.74 to a highly modified camelid CD16 (FC?RIII)-specific scFv fragment . The latter binds to NK cells, while the former to TNBC cells. These novel hybrid molecules which are referred to as IL-15 TriKEs enhance the ability of NK cells to kill tumor cells with limited -if any- damage to normal cells. iii) To restore the susceptibility of TNBC cells to immune lysis we combine IL-15 TriKEs with the small molecule LDE225, an inhibitor of the SHH pathway, since the activation of this pathway up-regulates the level of anti-apoptotic molecules. iv) To enhance the anti-tumor activity of NK cells we disrupt the PD-1/PD-L1 axis with an anti-PD-L1 mAb. We will test the hypothesis that NK cells in combination with IL-15 TriKEs, LDE225 and anti-PD-L1 mAb are effective in eradicating both differentiated TNBC cells and TNBC CICs, both in vitro and in vivo. As a result, TNBC disease recurrence and metastatic spread will be suppressed. Because of the limited budget the proposed studies will be performed only with TNBC cell lines. In future studies the potential clinical significance of the results obtained with cell lines will be assessed with patient derived xenografts. This information will then be translated to a clinical setting in a Phase I clinical trial.